Acta Protozoologica 2008 48/2
Jagiellonian University Kraków, Poland
© Jagiellonian University
Acta Protozool. (2008) 47: 161–171
Acta
Protozoologica
A Molecular Survey of Paramecium dodecaurelia (Ciliophora, Protozoa)
Strains from a Single Pond
Ewa PRZYBOŚ, Sebastian TARCZ and Marta SURMACZ
Institute of Systematics and Evolution of Animals, Polish Academy of Sciences, Kraków, Poland
Summary: The aim of the study was to investigate if strains of P. dodeaurelia collected from different sampling points situated around
the single habitat (a pond in Dr Jordan’s Park, Kraków, Poland) and originated from successive seasons may be characterized by different
haplotypes. So, we expected the spatial and seasonal differentiation of P. dodecaurelia haplotypes originated from a single habitat, as the
studied species is characterized by high intra-specific polymorphism. Fragments of 5’LSU rDNA (349 bp long) and COI mtDNA (309 bp
long) of 18 strains of Paramecium dodecaurelia were compared, five of these came from the studied pond (strains A, B, C1, C2, C3) and others from our collection originating from Europe (Italy, Germany, Russia, Ukraine), Asia (Kazakhstan, Russia Eastern Siberia [RS], Japan),
island of Tenerife, mainland USA and Hawaii.The following species were used as outgroups: a standard strain of P. pentaurelia (87, from
Pennsylvania, USA), a representative of the P. aurelia complex showing no intraspecific differentiation; and P. caudatum, a representative
of a different Paramecium morphospecies. A comparison of the distance matrix did not show any variability among strains from Kraków.
The same haplotype appeared in several strains from Europe (RM, RV, RY, UV, IE, Poland), Asia (KA, RS), and Tenerife (TE). A different haplotype was detected in the other European strains from Germany and Italy (TR). Strains from USA, Japan, and Hawaii were more
distant from other P. dodecaurelia strains. Polymorphism of P. dodecaurelia was confirmed, however, no correlation was found between
geographical origin and genetic distance of strains. The strain from Trento, Italy of P. dodecaurelia revealed the same haplotype as strain
87 of P. pentaurelia, in spite of sexual isolation of both species. Perhaps, speciation of P. aurelia complex proceeds by a different way than
differentiation of studied DNA fragments and relationships of species of the P. aurelia complex is more complicated that was expected. It
may be connected with the origin of the P. aurelia complex. The hypothesis on the origin of the complex, proposed by Aury et al. (2006),
is presented in discussion.
Key words: Paramecium dodecaurelia, rDNA, COI mtDNA, genetic differentiation, one pond, intraspecific differentiation, relationships
of Paramecium aurelia species.
INTRODUCTION
Intraspecific molecular polymorphism was revealed
within the P. aurelia species first by RAPD analysis
Corresponding Author: S. Tarcz, Institute of Systematics and
Evolution of Animals, Polish Academy of Sciences, Sławkowska
17, 31-016 Kraków, Poland; tel.: 00 4812-422-80-00; Fax: 00 4812
422 42 94; E-mail: [email protected]
(Stoeck and Smidt 1998; Stoeck et al. 1998, 2000;
Przyboś and Tarcz 2005; Przyboś et al. 2005, 2006b,
2007a). Later, the studies were concerned mainly on
species showing high polymorphism, such as P. dodecaurelia and P. novaurelia. They were carried out by
sequencing of rRNA and COI mtDNA fragments (Tarcz
et al. 2006, Przyboś et al. 2007b, Przyboś et al. 2008).
According to these studies, the intraspecific polymorphism of P. dodecaurelia was as high as that between
different species of the P. aurelia complex.
Acta Protozoologica 2008 48/2
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© Jagiellonian University
162 E. Przyboś et al.
Stoeck et al. (1998, 2000) suggested that intraspecific
polymorphism may be associated with a degree of inbreeding which is characteristic (Sonneborn 1957, Sonneborn 1975, Landis 1986) for species of the complex.
Landis (1986) proposed to characterize species of the P.
aurelia complex by various combinations of ecology,
breeding systems and genetic variation and considered
that “species of the P. aurelia complex are comparative
specialists, ... having an optimized genotype maintained
by an inbreeding strategy”. However, according to Foissner (2006) and Foissner et al. (2008) genetic and molecular bio-geographic studies are still sparse in ciliates”,
and according to Groben et al. (2007) molecular methods may bring new data for assessment of biodiversity
in a particular ecosystem. Finlay et al. (2006) supposed
that most free-living microbial eukaryote morphospecies
consist of multiple ecotypes with different genotypic and
phenotypic divergences in local niches (Finlay 2004),
which has consequences for quantifying “biodiversity.”
Paramecia appearing in one habitat (water body) might
occupy different niches characterized by various environmental features (bacteria, organic matter, temperature). Similarly, studies on the distribution of the North
American tetrahymena species showed the possibility of
their ecological specialization (Nanney 2004).
A survey of the population structure of a protozoan
was carried out by Barth et al. (2006) who discovered
different mitochondrial haplotypes of P. caudatum in
one pond. Seven clonal lineages of P. caudatum were
studied and two mitochondrial haplotypes were obtained differing in three nucleotide positions. Earlier,
Kusch (1998) found four “genotypes” in Stentor coeruleus populations by RAPD. Recently, Barth et al.
(2008) also found mitochondrial haplotype diversity of
Coleps sp. (Ciliophora: Prostomatida) in a community
from one lake, as 11 mitochondrial haplotypes in Coleps spetai and 9 in Coleps hirtus hirtus were revealed.
The aim of the present study was to investigate if paramecia of P. dodecaurelia with different haplotypes may
exist in a single habitat (pond) and if populations of paramecia originating from different seasons are characterized by different haplotypes. The samples were collected
from six sampling points situated around the pond during two successive seasons. We expected the spatial and
seasonal differentiation of P. dodecaurelia haplotypes
originated from that habitat (pond), as high intra-specific
polymorphism of the studied species has been revealed
previously. Fragments of rRNA and mtDNA of P. dodecaurelia strains originating from different localities in Europe, Asia and Northern America were compared and of
P. pentaurelia and P. caudatum, representing outgroup.
MATERIAL AND METHODS
Material
The studied P. dodecaurelia strains originated from an artificial pond (constructed in 1957) situated in Dr. H. Jordan’s Park,
Kraków, Poland.
Procedures of paramecia collection. The routine procedure
of collecting and establishing clones from nature (cf Komala and
Przyboś 1984) was as follows: samples of water with plankton and
plant remnants were taken from the surface of littoral waters. From
each sampling point 10 samples (tubes) were taken with about 20 ml
of water. The pH and the temperature of the water were measured
simultaneously, as well as the ambient temperature. A day after collection, all paramecia (3–8 individuals) found in the particular tubes
(samples) were separately isolated from each sample and clones were
established. A small amount of medium was added to the samples in
which no paramecia were found and after a week the samples were
reexamined. The second examination revealed only P. caudatum in
several “blooming” samples. The collecting was done by M. Surmacz
in May 2006, May 2007, and in October 2007. Each time the samples
were collected from six sampling points (s.p.) situated around the
pond (marked by black dots in Fig. 1). In May 2006 paramecia were
found only in one sampling point, designated A (the established strain
was designated as the A strain; pH 4.8, water temperature 18°C, ambient temperature 24°C), in May 2007 paramecia were found in two
s.p. designated B and C (B strain and C strain; pH 5.0, water temperature 18°C, ambient temperature 20°C (Fig. 1). In case of the C
s.p., paramecia were found in three samples, so the established strains
C
N
2007
B
2007
A
2006
100 m.
Fig. 1. Sampling points (marked by dots) in the pond in Dr. Jordan’s
Park in Kraków, Poland. Points where paramecia were found designated by A, B, C.
Acta Protozoologica 2008 48/2
Jagiellonian University Kraków, Poland
© Jagiellonian University
Paramecium dodecaurelia from a single pond 163
were designated CI, CII, CIII. No paramecia were found in October
2007, which was perhaps connected with the chemical composition
of the water (pH 5.6, water temp. 9°C, ambient temp. 12°C).
METHODS
1. Culturing and identification of paramecia
Paramecia were cultured (in a medium made of dried
lettuce and distilled water, inoculated with Enterobacter aerogenes), and identified according to the methods
of Sonneborn (1970). Investigated clones matured for
conjugation were mated with the reactive complementary mating types of the standard strains of a few species
of the P. aurelia complex morphologically similar to the
investigated ones. The following standard strains were
used: strain 90 from Pennsylvania, USA standard of
P. primaurelia; strain 87 from Pennsylvania, USA; standard strain of P. pentaurelia; strain 246 from Mississippi, USA, standard strain of Paramecium dodecaurelia.
2. Identification of morphospecies
Paramecia were analyzed on slides stained using
aceto-carmine (Sonneborn 1970) and identified to the
particular species of Paramecium genus by the type and
number of micronuclei (Vivier 1974). All paramecia
from the P. aurelia complex have a nuclear apparatus
with two vesicular micronuclei, while species belonging to P. caudatum contain one compact micronucleus.
3. Strain crosses
In the intra and inter-strain crosses, the F1 generation
was obtained by conjugation and F2 by autogamy (using
the method of daily isolation lines). The occurrence of the
desired stage of autogamy (specimens at the stage of two
macronuclear anlagen) was examined on preparations
stained with aceto-carmine. Survival of clones in both
generations was estimated as percentages. According to
Chen (1956), clones can be considered as surviving after passing 6–7 fissions during 72 hours after separation
of partners of conjugation or postautogamous caryonids.
The methods were described in detail in Przyboś (1975).
4. Methods used in molecular studies
Paramecium genomic DNA was isolated (200 μl of
cell culture (about 200 cells) was used for DNA extraction) from vegetative cells at the end of the exponential
phase using the Qiamp DNA Kit (Qiagen™, Germany)
as described by Przyboś et al. (2003). The strains used
in the present study are listed in Table 1.
a. Amplification of ribosomal DNA (rDNA)
The primers used to PCR amplify the 5’ end of
a fragment of LSU rDNA (450bp), they are listed in Table 2. The forward primer was constructed using Oligoanalyzer 3.0 (http://scitools.idtdna.com/analyzer/). The
reverse primer – LR6, is a universal eukaryotic primer
for amplification of LSU rDNA fragment. (http://www.
biology.duke.edu/fungi/mycolab/primers.htm). PCR
amplification was carried out in a final volume of 30 μl
containing: 2 μl of DNA, 1.5 U Taq-Polymerase (Qiagen, Germany), 0.6 μl 10mM of each primer, 10x PCR
buffer, 0.6 μl of 10mM dNTPs in a T-personal thermocycler ™ (Biometra GmbH, Germany). The amplification protocol consisted of initial denaturation at 94°C,
followed by 34 cycles of denaturation at 94°C for 45 s,
annealing at 50°C for 60 s, and extension at 72°C for 60
s, with final extension at 72°C for 5 min. After amplification the PCR products were electrophoresed in 1%
agarose gels for 45 min at 85 V with a DNA molecular
weight marker (VI ™ Roche, France).
b. Amplification of gene fragments of mitochodrial
cytochrome oxidase (COI)
To amplify the CO I region (880bp) of mitochonrial
DNA, Cox_L and Cox_H primers were used (according
to Barth et al. 2006). PCR amplification was carried out
in the same volume as in the case of the rDNA region (see
above), and the protocol followed Barth et al. (2006).
After amplification, the PCR products were electrophoresed in 1% agarose gels for 45 min at 85 V with a DNA
molecular weight marker (VI ™ Roche, France).
c. Purification and sequencing
30 μl of each PCR product was separated on a 1.8%
agarose gel (100 V/60 min). Then, the band representing the examined fragment was cut out and purified
using by the Qiaquick Gel Extraction Kit™ (Qiagen).
Cycle sequencing was done in both directions using the
BigDye Terminator v3.1™ chemistry (Applied Biosystems, USA). Sequencing products were precipitated using Ex Terminator™ (A&A Biotechnology, Poland) and
separated on an ABI PRISM 377 DNA Sequencer™
(Applied Biosystems, USA).
d. Data analysis
Sequences were examined using Chromas Pro (Technelysium™, Australia) to evaluate and correct chromatograms. Alignment and consensus of the studied
sequences was performed using ClustalW (Thompson
et al. 1994) in the BioEdit program (Hall 1999). Trees
were constructed for the studied fragments in Mega
Acta Protozoologica 2008 48/2
Jagiellonian University Kraków, Poland
© Jagiellonian University
164 E. Przyboś et al.
Table 1. Paramecium dodecaurelia strains used in molecular studies.
Species
Strain
Geographical origin
designation
Reference
P. dodecaurelia
246
Sonneborn 1974
USA, southern state
Accesion number
5’LSU rDNA
COI mtDNA
DQ207369
EU086108
P. dodecaurelia
A
Poland, Kraków, Jordan’s Park
present paper
EU086120*
EU086111*
P. dodecaurelia
B
Poland, Kraków, Jordan’s Park
present paper
EU086120*
EU086111*
P. dodecaurelia
C1
Poland, Kraków, Jordan’s Park
present paper
EU086120*
EU086111*
P. dodecaurelia
C2
Poland, Kraków, Jordan’s Park
present paper
EU086120*
EU086111*
P. dodecaurelia
C3
Poland, Kraków, Jordan’s Park
present paper
EU086120*
EU086111*
P. dodecaurelia
G
Germany, Münster
Przyboś and Fokin 2003b
DQ 207370
EU487004
P. dodecaurelia
HHS
Hawai, Honolulu
Przyboś and Fokin 2003a
DQ 207371
EU487005
P. dodecaurelia
IE
Italy, Elbe Island
Przyboś and Fokin 2003b
DQ207372
EU086109
P. dodecaurelia
JU
Japan, Honshu island, Ube city
Przyboś et al. 2003
DQ 207373
EU487006
P. dodecaurelia
KA
Kazakhstan, Aral region, Sarbas
Przyboś et al. 2008
EU086119
EU086110
P. dodecaurelia
RM
Russia, Myshkin
Przyboś et al. 2008
EU086121
EU086112
P. dodecaurelia
RS
Russia, Ułan Ude, Selenga River
Przyboś et al. 2008
EU086122
EU086113
P. dodecaurelia
RV
Russia, Vologda region
Przyboś et. al. 2008
EU086123
EU086114
P. dodecaurelia
RY
Russia, Yaroslavl region
Przyboś et. al. 2008
EU086124
EU086115
P. dodecaurelia
UV
Ukraine, Vinnitsa
Przyboś et al. 2008
EU086126
EU086117
P. dodecaurelia
TE
Canary Is., Tenerife, Puerto de la Cruz
Przyboś et al. 2008
EU086125
EU086116
P. dodecaurelia
TR
Italy, Trento
Przyboś et al. 2005
DQ 207374
EU487007
P. pentaurelia
87
USA, Pennsylvania
Sonneborn 1974
EU086127
EU086118
P. caudatum
Pc
Cyprus, Akamas
Przyboś unpublished
DQ207375
DQ837977
* there were no differences between strains so we have used accession numbers of strain PK (data from Przyboś et al. 2008a).
Table 2. Primers used in this study.
Amplified region
5’LSU rDNA
CO I mtDNA
Primer
Sequence 5’–3’
References
LSU_F
5’-CCCGTATTTGGTTAGGACT-3’
TARCZ et al. 2006
LR6
5’-CGCCAGTTCTGCTTACC-3’
Universal eukaryotic primer*
CoxL11058
5’-TGATTAGACTAGAGATGGC-3’
BARTH et al. 2006
CoxH10176
5’-GAAGTTTGTCAGTGTCTATCC-3’
BARTH et al. 2006
* http://www.biology.duke.edu/fungi/mycolab/primers.htm
version 3.1 (Kumar et al. 2004), using the NeighborJoining method (NJ) (Saitou and Nei 1987) and Maximum Parsimony (MP) (Nei and Kumar 2000). The NJ
analysis was performed using a Kimura 2-parameter
correction model (Kimura 1980) and Jukes-Cantor
method (Jukes and Cantor 1969) by bootstrapping with
1000 replicates (Felsenstein 1985). The MP analysis
was evaluated with Min-mini heuristic parameter (level = 2) and bootstrapping with 1000 replicates.
Acta Protozoologica 2008 48/2
Jagiellonian University Kraków, Poland
© Jagiellonian University
Paramecium dodecaurelia from a single pond 165
RESULTS
Strain identification based on mating reaction
The strains of paramecia established in 2006
(A strain, 3 clones, all originating from one sample) and
2007 (strain B, 3 clones originating from one sample;
strain C, 7 clones originating from sample I, 8 clones
from sample II, 5 clones from sample III) were identified as P. dodecaurelia on the basis of strong conjugation with the standard strain of this species. A high
percentage of surviving clones was observed in F1 and
F2 generations of all inter-strain crosses of the strains
(Table 3) within P. dodecaurelia.
Analysis of the fragment of the large subunit of ribosomal DNA (5’ LSU rDNA)
Analysis of the mitochondrial cytochrome oxidase
gene fragment (COI mtDNA)
Fragments of COI mtDNA (309 bp) of 18 strains
of P. dodecaurelia, and single strains of P. pentaurelia
(87) and P. caudatum, were sequenced and compared,
Germany, Münster (G)
Russia, Selenga (RS)
Russia, Myshkin (RM)
Russia, Vologda (RV)
Russia, Yaroslavl (RY)
Kazakhstan, Sarbas (KA)
100/99 Ukraine, Vinnitsa (UV)
Italy, Elbe Island (IE)
Tenerife (TE)
Poland, Kraków (B)
Poland, Kraków (C1)
Poland, Kraków (C2)
Jordan’s Park
Poland, Kraków (A)
91/78
P. dodecaurelia
Fragments of 5’ LSU rDNA (349 bp) of 18 strains of
Paramecium dodecaurelia were compared, five (A, B,
C1, C2, C3) of them from the studied pond in Kraków,
and others from our collection originating from Europe
(Italy, Germany, Russia, Ukraine), Asia (Kazakhstan,
Russia Eastern Siberia [RS], Japan), Tenerife, mainland
USA and Hawaii. We expected to find high intraspecific
differentiation within P. dodecaurelia connected with
geographic origin of strains as well as with spatial and
seasonal differentiation of paramecia from the studied
pond in Kraków. For outgroups, a standard strain (87,
from USA, Pennsylvania) of P. pentaurelia was used
as a representative of species of the P. aurelia complex
showing no intraspecific differentiation, which was revealed by RAPD previous studies (Przyboś et al. 2005,
2007a), and P. caudatum as a representative of a different morphospecies in Paramecium genus (Fig. 2).
On the basis of analysis of 40 variable nucleotide
positions (18 informative sites), it was revealed that
the studied strains were characterized by seven haplotypes (when all studied strains, i.e. including P. dodecaurelia, P. pentaurelia and P. caudatum were used).
All polymorphic sites were localized within the length
of 190 bp of the studied fragment. The mean variability (genetic distance) of the 5’LSU rRNA fragment of
all Paramecium strains was at the level of 2.4%, and
1.4% within P. dodecaurelia, according to the Kimura
and Jukes-Cantor models. Comparison of the distance
matrix (Table 4) did not show any variability among
strains from Kraków. The same haplotype appeared
in several strains from Europe (RM, RV, RY, UV, IE,
Poland), Asia (KA, RS), and Tenerife (TE). A different
haplotype was observed in the other European strains
from Germany and Italy (TR). Notably, a similar haplotype was observed in strains from distant stands such as
Tenerife, Europe (several locations), and Asia (RS), and
on the other hand the same haplotype was characteristic for strains from Trento, Italy of P. dodecaurelia and
strain 87 (from USA) belonging to different species, i.e.
P. pentaurelia and included as an outgroup. The other
strains of P. dodecaurelia (246 from mainland USA, Japan and Hawaii) had three different haplotypes.
The tree (based on NJ and MP analyses) showed that the
P. dodecaurelia strains are divided into four clusters and
that the P. caudatum haplotype is distant (an outgroup).
Poland, Kraków (C3)
90/88
P. dodecaurelia Italy, Trento (TR)
65/87 P. penturelia (87)
P. dodecaurelia USA (246)
78/83 P. dodecaurelia Japan, Honsiu (JU)
P. dodecaurelia Hawai, Honolulu (HHS)
P. caudatum
0.005
Fig. 2. Tree constructed for 18 strains of P. dodecaurelia (P. pentaurelia strain and P. caudatum strain as outgroup), based on a comparison
of sequences from 5’ LSU rDNA fragment using the NJ (neighbor
joining) method (with the application of the Kimura two-parameter
and Jukes-Cantor correction model) and MP (maximum parsimony)
analysis. Bootstrap values are presented as percentages (K2P/J-C/MP)
for 1000 replicates. All positions containing gaps and missing data
were eliminated from the dataset. There were a total of 349 positions
in the final dataset. Analyses were conducted in MEGA 3.1.
Acta Protozoologica 2008 48/2
Jagiellonian University Kraków, Poland
© Jagiellonian University
166 E. Przyboś et al.
Table 3. Mean percentage of surviving hybrid clones in crosses of the Paramecium dodecaurelia strains.
Strain
F1 (by conjugation)
F2 (by autogamy)
94
76
*A (Kraków, Poland) x RV (Vologda, Russia)
100
94
A (Kraków, Poland) x 246 (Missisipi, USA)
100
96
B (Kraków, Poland) x 246 (Missisipi, USA)
100
92
B (Kraków, Poland) x RV (Vologda, Russia)
100
92
CI (Kraków, Poland) x 246 (Missisipi, USA)
90
90
CII (Kraków, Poland) x 246 (Missisipi, USA)
94
90
CIII (Kraków, Poland) x 246 (Missisipi, USA)
96
100
*A (Kraków, Poland x KA (Kazahstan)
* Data from Przyboś et al. 2008a (Table 3).
Russia, Myshkin (RM)
Russia, Vologda (RV)
Russia, Yaroslavl (RY)
Kazakhstan (KA)
*/*/56
Ukraine, Vinnitsa (UV)
Poland, Kraków (B)
54/58/77
Poland, Kraków (C1)
Poland, Kraków (C2)
79/78/77
100/100/99
*/*/70
Jordan’s Park
Poland, Kraków (A)
P. dodecaurelia
the latter two strains were used as outgroups (Fig. 3).
The fragment of 309 bp was used in the present studies
as only such length was clear enough for analyses for
investigated strains and showed intraspecific differences in satisfactory way. The mtDNA fragment was more
variable than the rDNA fragment, as 10 haplotypes were
revealed in the studied material. 106 variable nucleotide positions were observed with substitutions, 70 of
these were informative. Also several variable sites were
found: 20 with three nucleotide variants and 5 with four
nucleotide variants. It is worth to mention that many of
substitutions are silent mutations as taking place at the
third base of a codon.
Comparison of the distance matrix (Table 5) shows
that the mean variability (genetic distance) of the studied strains was at the level of 9.7% (higher than in the
case of rDNA) when all strains were compared (P. dodecaurelia, P. pentaurelia and P. caudatum), and 6.7%
within P. dodecaurelia.
In spite of higher variability of mtDNA, all strains
from Kraków show the same haplotype, as well as
strains from Russia (RM, RV, RY), Kazakhstan and
Ukraine. Strains from Germany and Selenga, Russia
showed slightly different haplotypes, as well as strains
from Italy, IE and Tenerife. Strains from USA, Japan,
and Hawaii were more distant from other P. dodecaurelia strains, similarly as in the analysis of 5’ LSU rDNA.
Again, the strain from Trento, Italy of P. dodecaurelia revealed the same haplotype as strain 87 of P. pentaurelia.
The relationships of species within the P. aurelia complex is more complicated that we expected. The attempt
of possible explanation is presented in discussion.Fig. 3
Poland, Kraków (C3)
Germany, Münster (G)
Russia, Selenga (RS)
Italy, Elbe Island (IE)
83/86/75
Spain, Tenerife (TE)
P. dodecaurelia Italy, Trento (TR)
*/*/*
100/100/100 P. penturelia (87)
P. dodecaurelia USA (246)
*/*/*
P. dodecaurelia Japan, Honsiu (JU)
P. dodecaurelia Hawai, Honolulu (HHS)
P. caudatum
0.02
Fig. 3. Tree constructed for 18 strains of P. dodecaurelia (P. pentaurelia strain and P. caudatum strain as outgroup), based on a comparison of sequences from COI mtDNA fragment using the NJ (neighbor
joining) method (with the application of the Kimura two-parameter
and Jukes-Cantor correction model) and MP (maximum parsimony)
analysis. Bootstrap values are presented as percentages (K2P/J-C/MP)
for 1000 replicates. In the case of bootstrap values less than 50, the
asterisk appears. All positions containing gaps and missing data were
eliminated from the dataset. There were a total of 309 positions in the
final dataset. Analyses were conducted in MEGA 3.1.
DISCUSSION
The most characteristic feature of both trees (obtained by NJ and MP methods and with similar topography) (Figs 2 and 3) is one clade with the majority of
the studied P. dodecaurelia strains including strains from
Kraków. The strains from Jordans’ Park, Kraków repre-
Acta Protozoologica 2008 48/2
Jagiellonian University Kraków, Poland
© Jagiellonian University
Paramecium dodecaurelia from a single pond 167
Table 4. Distance matrix presenting the number of base substitutions in Paramecium dodecaurelia, P. pentaurelia and P. caudatum strains,
based on analyses of 5’LSU rDNA sequences. Analyses were conducted using the Kimura 2-parameter method (lower-left) and Jukes-Cantor method (upper-right).
5’LSU
P.dodec, 246
P.dodec, IE
P.dodec, G
P.dodec, HHS
P.dodec, JU
P.dodec, TR
P.dodec, KA
P.dodec, RM
P.dodec, RS
P.dodec, RV
P.dodec, RY
P.dodec, UV
P.dodec, TE
P.dodec, A
P.dodec, B
P.dodec, C I
P.dodec, C II
P.dodec, C III
P.pent.
P.caud.
P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec,
P.pent P.caud.
246
IE
G
HHS
JU
TR
KA
RM
RS
RV
RY
UV
TE
A
B
CI
C II
C III
0.032
0.032
0.035
0.026
0.003
0.017
0.032
0.032
0.032
0.032
0.032
0.032
0.032
0.032
0.032
0.032
0.032
0.032
0.017
0.092
0.003
0.054
0.029
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.020
0.103
0.035
0.003
0.057
0.032
0.023
0.003
0.003
0.003
0.003
0.003
0.003
0.003
0.003
0.003
0.003
0.003
0.003
0.023
0.106
0.026
0.053
0.057
0.029
0.032
0.054
0.054
0.054
0.054
0.054
0.054
0.054
0.054
0.054
0.054
0.054
0.054
0.032
0.076
0.003
0.029
0.032
0.029
0.014
0.029
0.029
0.029
0.029
0.029
0.029
0.029
0.029
0.029
0.029
0.029
0.029
0.014
0.089
0.017
0.020
0.023
0.032
0.014
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.000
0.086
0.032
0.000
0.003
0.053
0.029
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.020
0.103
0.032
0.000
0.003
0.053
0.029
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.020
0.103
0.032
0.000
0.003
0.053
0.029
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.020
0.103
0.032
0.000
0.003
0.053
0.029
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.020
0.103
0.032
0.000
0.003
0.053
0.029
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.020
0.103
0.032
0.000
0.003
0.053
0.029
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.020
0.103
0.032
0.000
0.003
0.053
0.029
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.020
0.103
0.032
0.000
0.003
0.053
0.029
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.020
0.103
0.032
0.000
0.003
0.053
0.029
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.020
0.103
0.032
0.000
0.003
0.053
0.029
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.020
0.103
0.032
0.000
0.003
0.053
0.029
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.020
0.103
0.032
0.000
0.003
0.053
0.029
0.020
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.000
0.017
0.020
0.023
0.032
0.014
0.000
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.020
0.103 0.086
0.091
0.101
0.104
0.075
0.088
0.085
0.101
0.101
0.101
0.101
0.101
0.101
0.101
0.101
0.101
0.101
0.101
0.101
0.085
Table 5. Distance matrix presenting the number of base substitutions per site from analyses of P. dodecaurelia, P. pentaurelia and P. caudatum strains based on analyses COI mtDNA sequences. Analyses were conducted using the Kimura 2-parameter method (lower left) and
Jukes-Cantor method (upper right).
COI
P.dodec, 246
P.dodec, IE
P.dodec, G
P.dodec, HHS
P.dodec, JU
P.dodec, TR
P.dodec, KA
P.dodec, RM
P.dodec, RS
P.dodec, RV
P.dodec, RY
P.dodec, UV
P.dodec, TE
P.dodec, A
P.dodec, B
P.dodec, C I
P.dodec, C II
P.dodec, C III
P.pent
P.caud.
P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec, P.dodec,
P.pent P.caud.
246
IE
G
HHS
JU
TR
KA
RM
RS
RV
RY
UV
TE
A
B
CI
C II
C III
0.154
0.157
0.166
0.174
0.151
0.167
0.162
0.162
0.166
0.162
0.162
0.162
0.161
0.162
0.162
0.162
0.162
0.162
0.167
0.296
0.010
0.170
0.144
0.150
0.010
0.010
0.013
0.010
0.010
0.010
0.003
0.010
0.010
0.010
0.010
0.010
0.150
0.263
0.162
0.010
0.165
0.148
0.162
0.003
0.003
0.013
0.003
0.003
0.003
0.013
0.003
0.003
0.003
0.003
0.003
0.162
0.268
0.170
0.166
0.162
0.161
0.202
0.165
0.165
0.178
0.165
0.165
0.165
0.174
0.165
0.165
0.165
0.165
0.165
0.202
0.260
0.146
0.142
0.146
0.158
0.170
0.149
0.149
0.149
0.149
0.149
0.149
0.148
0.149
0.149
0.149
0.149
0.149
0.170
0.275
0.162
0.146
0.158
0.195
0.166
0.163
0.163
0.167
0.163
0.163
0.163
0.154
0.163
0.163
0.163
0.163
0.163
0.000
0.274
0.158
0.010
0.003
0.162
0.146
0.158
0.000
0.010
0.000
0.000
0.000
0.013
0.000
0.000
0.000
0.000
0.000
0.163
0.269
0.158
0.010
0.003
0.162
0.146
0.158
0.000
0.010
0.000
0.000
0.000
0.013
0.000
0.000
0.000
0.000
0.000
0.163
0.269
0.162
0.013
0.013
0.174
0.146
0.162
0.010
0.010
0.010
0.010
0.010
0.016
0.010
0.010
0.010
0.010
0.010
0.167
0.284
sent only one haplotype, which may be caused by a single founding population which settled the pond in 1957.
The tree based on the analysis of sequences of
5’LSU rDNA shows only slight variability (strain from
Germany) of this large clade.
0.158
0.010
0.003
0.162
0.146
0.158
0.000
0.000
0.010
0.000
0.000
0.013
0.000
0.000
0.000
0.000
0.000
0.163
0.269
0.158
0.010
0.003
0.162
0.146
0.158
0.000
0.000
0.010
0.000
0.000
0.013
0.000
0.000
0.000
0.000
0.000
0.163
0.269
0.158 0.158
0.010 0.003
0.003 0.013
0.162 0.170
0.146 0.146
0.158 0.150
0.000 0.013
0.000 0.013
0.010 0.016
0.000 0.013
0.000 0.013
0.013
0.013
0.000 0.013
0.000 0.013
0.000 0.013
0.000 0.013
0.000 0.013
0.163 0.154
0.269 0.268
0.158 0.158
0.010 0.010
0.003 0.003
0.162 0.162
0.146 0.146
0.158 0.158
0.000 0.000
0.000 0.000
0.010 0.010
0.000 0.000
0.000 0.000
0.000 0.000
0.013 0.013
0.000
0.000
0.000 0.000
0.000 0.000
0.000 0.000
0.163 0.163
0.269 0.269
0.158
0.010
0.003
0.162
0.146
0.158
0.000
0.000
0.010
0.000
0.000
0.000
0.013
0.000
0.000
0.000
0.000
0.163
0.269
0.158
0.010
0.003
0.162
0.146
0.158
0.000
0.000
0.010
0.000
0.000
0.000
0.013
0.000
0.000
0.000
0.000
0.163
0.269
0.158
0.010
0.003
0.162
0.146
0.158
0.000
0.000
0.010
0.000
0.000
0.000
0.013
0.000
0.000
0.000
0.000
0.162
0.146
0.158
0.195
0.166
0.000
0.158
0.158
0.162
0.158
0.158
0.158
0.150
0.158
0.158
0.158
0.158
0.158
0.163
0.269 0.274
0.284
0.256
0.260
0.251
0.265
0.265
0.260
0.260
0.274
0.260
0.260
0.260
0.260
0.260
0.260
0.260
0.260
0.260
0.265
Greater variability of this group of strains (clade)
can be seen in the tree constructed on the basis of COI
mtDNA sequences, as strains from Italy (IE) and Tenerife (TE) now appear in different branches, also strains
from Germany and Selenga, Russia (Eastern Siberia)
Acta Protozoologica 2008 48/2
Jagiellonian University Kraków, Poland
© Jagiellonian University
168 E. Przyboś et al.
show different relationships to the other strains. The
P. pentaurelia strain forms a closely related clade with
the strain of P. dodecaurelia from Trento, Italy and is
distant to the other European strains.
In both trees, the strain from Hawaii appears as
a distant branch, which may be due to the geographical
distance (isolation) from other strains. Similarly, strains
from USA and Japan form one clade, distant from other
strains, and the P. caudatum clade is clearly distant.
A very puzzling feature appearing in both trees is
the close relationship of different species of the P. aurelia complex, as between strain 87 of P. pentaurelia and
the strain from Italy (TR) of P. dodecaurelia. A similar
situation was also observed among some P. novaurelia
strains (Tarcz 2007), as some were related with P. biaurelia and P. sexaurelia strains. Also in the paper (Barth
et al. unpublished) some strains representing different
species of the P. aurelia complex (P. tetraurelia, P. sixaurelia, P. octaurelia and P. decaurelia) showed similar
cytochrome b sequences. Perhaps such molecular relationship of strains representing different species of the
P. aurelia complex is allowing to suppose that molecular similarity of the studied fragment is older than sexual
isolation of species. Perhaps, speciation of P. aurelia
complex proceeds by a different way than differentiation of studied DNA fragments. The molecular results,
obtained at present, may recall the problem of origin
of species of the P. aurelia complex. Aury et al. (2006)
supposed that most of P. tetraurelia genes arose through
three successive whole-genome duplications. They were
followed by reciprocal silencing of duplicated genes in
different populations which caused later isolation of
them and explosive radiation of species. According to
Aury et al. (2006) “the most recent duplication coincides with an explosion of speciation events that gave
rise to the P. aurelia complex of 15 sibling species.”
Investigations concerning P. dodecaurelia are of interest because strains of this species show high polymorphism (Przyboś et al. 2005, 2008a; Tarcz et al.
2006) which may be connected with extreme inbreeding, characteristic for the species. Intraspecific polymorphism within P. dodecaurelia was already shown
by RAPD studies (Przyboś et al. 2005), as well as by
complex analyses including RAPD, RFLP, and ARDRA
(Przyboś et al. 2007a). Also sequencing of a fragment
of histone H4 carried out in standard strains representing 11 species of the P. aurelia complex (Przyboś et al.
2006a) showed the isolated position of P. dodecaurelia
within a tree constructed for 11 species of the complex,
as well as analysis of sequences of the hsp 70 protein
(Hori et al. 2006). Similarly, studies of the macronuclear genomes of species of the P. aurelia complex by
pulsed-field gel electrophoresis (PFGE) showed exceptional features of P. dodecaurelia, as each species of
the complex can be characterized by an individual electrokaryotype for all strains of the species, only in the
case of this species, each strain has a distinctive PFGE
profile (Potekhin et al. 2005).
Preliminary sequencing of rDNA fragments (3’ end of
rRNA – ITS1 (210bp) and 5’ end of LSU rRNA (350bp)
in P. dodecaurelia, P. tredecaurelia and P. quadecaurelia (Tarcz et al. 2006) showed that intraspecific diversity within P. dodecaurelia was as high as that between
different species of the P. aurelia complex. In P. dodecaurelia (strains originating from remote collecting
sites in Europe, Japan, mainland USA and Hawaii) six
polymorphic sites were found in a fragment of rRNA
at the 3’ end of SSU rRNA– ITS1, only one site differentiating strains of P. tredecaurelia, and no changes
in P. quadecaurelia. Similarly, at the 5’ end of LSU
rRNA several polymorphisms characteristic for the
studied strains of P. dodecaurelia were observed, three
in P. tredecaurelia strains, and one in P. quadecaurelia
strains. Nanney et al. (1998) compared sequence differences in a variable 23S rRNA domain among several species of the P. aurelia complex and found that
pairs of species are separated by few changes. In turn,
Strüder-Kypke et al. (2000a, b) with application of SSU
rRNA gene sequences discovered that P. primaurelia
and P. tetraurelia differed by five nucleotides from each
other. Hori et al. (2006) found that nonsynonymous
substitutions were frequent in the Hsp70 gene in some
species of the P. aurelia complex, i.e. P. triaurelia,
P. septaurelia, P. dodecaurelia, where P. novaurelia,
P. tredecaurelia and P. quadecaurelia have one nonsynonymous substitution in the same position.
Molecular studies (RAPD and sequencing of LSU
rRNA and COI mt DNA gene fragments) of P. dodecaurelia strains originating from new stands of the species,
i.e. from Russia, Ukraine, Kazahstan, Poland and Tenerife, revealed intraspecific polymorphism as a difference between the strain from the USA and other strains
from different regions of the Palearctic. The genetic distance between P. dodecaurelia strains was at the level of
3.2%, according to Kimura and Jukes-Cantor method,
when gene fragment of 5’LSU rDNA was analyzed, and
4% (Kimura model) and 5% (Jukes-Cantor model) in
the case of COI mtDNA (Przyboś et al. 2008).
Acta Protozoologica 2008 48/2
Jagiellonian University Kraków, Poland
© Jagiellonian University
Paramecium dodecaurelia from a single pond 169
The strains analyzed in the present study showed
divergence of the 5’LSU rRNA fragment overall Paramecium strains (P. dodecaurelia, P. pentaurelia and
P. caudatum) at the level of 2.4% and 1.4% within
P. dodecaurelia, and at the level of 9.7% for all strains
in COI mtDNA, and 6.7% within P. dodecaurelia according to Kimura and Jukes-Cantor models. For comparison, intraspecific polymorphism within P. novaurelia was at the level of 2.4% in 3’SSU-ITS1 and 5.4% in
5’LSU fragments of rDNA and 13.9% divergence in the
case of ribosomal mtDNA (Przyboś et al. 2007b). Intraspecific differentiation within P. dodecaurelia revealed
by our previous studies (Tarcz et al. 2006; Przyboś et
al. 2007a; Przyboś et al. 2008) was a reason to investigate expected spatial and seasonal variability of P. dodecaurelia populations originated from a single habitat.
However, only one haplotype was found in this material
representing this species. Perhaps, future studies concerning similar problem should be carried out in natural
pond or lake with more differentiated microhabitat and
during several seasons.
It also seems that applied markers (5’LSU rDNA
and COI mtDNA) present variability of species of the
P. aurelia complex, as was shown by the present results
(intra-specific differentiation of P. dodecaurelia strains,
and close relationships of one P. dodecaurelia strain to
P. pentaurelia). However, the used markers did not discrimanate particular species of the complex. Mitochondrial COI sequences were also used in Tetrahymena
thermophila to examine population substructure (Simon
et al. 2008). It is worth mentioning that the fragment of
COI mtDNA investigated here has been recently used
as a “barcode of life,” i.e. a standard fragment of DNA
appearing in the majority of living organisms (Hebert et
al. 2003) enabling the analysis of phylogenetic relationships. It was also tested in Tetrahymena species (Lynn
and Strüder-Kypke 2006; Chantagsai and Lynn 2006;
Chantangsi et al. 2007) and recently (Barth et al. 2008)
a fragment of 547bp of the mitochondrial cob gene
was used in studies which revealed high mitochondrial
haplotype diversity of Coleps sp. (order Prostomatida)
from one lake (Lake Saidenbach, water reservoir of approximately 70 years old).
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Received on 14th March, 2008; revised version on 29th May, 2008;
accepted on 30th May, 2008